The long term goal of this MERIT extension continues to be to develop a comprehensive understanding of the regulation of cardiac excitation-contraction coupling (ECC) and contractile force, particularly with respect to myocyte Ca and Na regulation, which is central to the regulation of physiological function in the heart (both electrical and mechanical). Indeed, altered myocyte Ca transport is implicated in hypertrophy, heart failure and arrhythmias. This project period focuses on: 1) fundamental aspects of ECC and sarcoplasmic reticulum (SR) Ca handling, 2) regulation of Na/Ca exchange (NCX) and 3) dynamic regulation of calmodulin (CaM) in ventricular myocytes. Studies will use mainly isolated adult ventricular myocytes under voltage clamp, with confocal imaging &dynamic fluorescence measurements of [Ca]i, [Na]i, [CaM] and [Ca-CaM], complemented by biochemical/ molecular and computational studies. Genetically altered mice will also be used to test specific mechanisms. Aim 1 will address important fundamental ECC questions such as: critical mechanisms involved in Ca wave propagation, alternans and arrhythmias, quantitative analysis of mitochondrial Ca levels, and novel computer models incorporating myocyte data to test our quantitative understanding and fuel better integrative modeling. Aim 2 focuses on Na and Ca transport and will measure how NCX activates and deactivates under physiological conditions, how NCX association with annexin 5 modifies NCX function and elucidate how local subcellular [Na] gradients develop and influence cardiac function. Aim 3 will focus on CaM signaling in myocytes, obtaining new data from novel optical tools to assess translocation and partnering of CaM with targets, and activation states of CaM targets (CaMKII & calcineurin) in key subcellular domains (e.g. nucleus and sarcolemma) which influence systems from ion channels to transcriptional conntrol. A wide array of approaches will be used to answer key fundamental questions relevant to normal cardiac physiology and pathophysiology (e.g. of heart failure and arrhythmias). RELEVANCE (See instructions): Many aspects of cardiac function (mechanical and electrical) which go wrong in heart diseases, such as heart failure and arrhythmias, can be directly traced back to alterations in the way cardiac myocytes handle calcium, and how that influences the electrical and contractile properties of the heart. Here we will carry out careful quantitative studies to understand the fundamental workings of these systems critical to health and cardiac disease.